It is now common knowledge that stem cells in various epithelial tissues such as the basal layer of the epidermis, the bulge region of the hair follicle, and the crypt of the small intestine can be labeled following continuous administration with tritiated thymidine ([3H]TdR) at a critical stage of development. Initially, following such labeling procedures, all of the proliferating cells incorporate the label. However, following a chase period specific for each tissue, a very few cells in specific locations retain the label. These label-retaining cells have been identified as slowly cycling label-retaining cells (LRCs) in light microscopic autoradiographs. These observations are consistent with the hypothesis that these cells in the central position of the epidermal proliferative units (EPUs), in the bulge region of the hair follicles, and in cell position 4 of the intestinal crypts are stem cells. The reason usually given for the presence of LRCs in these tissues is that the LRCs divide more slowly than the other proliferating cells that rapidly dilute the DNA label by random DNA strand segregation. The problem with this explanation is that other evidence including that from mathematical models of cell renewal in intestinal or epidermal homeostasis strongly suggests that the LRCs have a cycle time approximately twice as long as the transit amplifying cells, and so should therefore reduce their label to background levels within four divisions. Hence, another explanation for label-retention by the stem cells must be found. Our objective is to determine the mechanism of label-retention by epidermal LRCs. We hypothesize that epidermal LRCs have a p53-dependent mechanism for selectively segregating their template DNA strands as well as a mechanism for protecting against DNA-replication-induced errors. We have two Specific Aims: 1) to determine whether stem cells in epidermis of p53+/+, p53+/-, and p53-/- mice retain an immortal DNA strand, and 2) to determine whether epidermal stem cells of p53+/+, p53+/-, and p53-/- have a mechanism for damage-induced apoptosis. The approach detailed in this proposal makes possible an answer to a compelling and fundamental problem in the biology of epithelial stem cells: whether keratinocyte stem cells in homeostasis segregate their template DNA strands and whether this is linked to altruistic cell suicide (apoptosis) in damaged stem cells. The research proposed here has critical implications not only for the structure and function of the epidermis, but also for the behavior of keratinocyte stem cells in chronic skin disease and cancer.